Biological Mechanisms of Dynamic White Matter Alterations

Abstract

Although the adult brain is perceived as largely
fixed, this concept is receiving increased scrutiny. One way in which the brain
can dynamically change in response to repeated stimuli is by altering the speed
of conductivity. Oligodendrocytes are a major cell type allowing for the
increased or reduced insulation of neuronal axons, through alterations in
myelination. One novel way to measure these changes is through the non-invasive
method of diffusion magnetic resonance imaging. This article will provide an
overview of the current state of research, and provide clues as to how
oligodendrocytes are mediating dynamic white matter alterations.

Introduction

Efficient connectivity is essential for the fast and
faithful delivery of signals across local and distal brain regions. White
matter axons with a sufficiently large axon diameter rely on myelin sheets from
nearby oligodendrocytes for saltatory conduction of their action potential
along the length of their axon. Although neuronal architecture is largely fixed
in adult mammals, in contrast, oligodendrocytes remain highly plastic
throughout adulthood and oligodendrocyte precursor cells (OPC) are the major
proliferating cell types in adult brains, constituting up to 9% of all cells in
the white matter (Dawson et al. 2003). The continued
plasticity into adulthood may be an important factor in continued motor
learning and high level cognitive functioning in humans (Wang and Young
2013).

Studies using in vivo diffusion
magnetic resonance imaging (MRI) methods have started to give insight into
dynamic alterations in white matter after training novel motor tasks such as
juggling (Scholz et al. 2009). Diffusion MRI as measured
with diffusion tensor imaging (DTI) offers a method of measuring integrity and
orientation of white matter in vivo (Figure 1). DTI allows for the
extraction of four types of measures that are selectively sensitive to
alterations in diffusivity that are thought to reflect specific biological
processes. Fractional anisotropy (FA) is a summary measure of structural
integrity. Mean diffusivity (MD) is an inverse measure of membrane density, and
is very similar for both gray and white matter, while being high for cerebral
spinal fluid. MD is sensitive to cellularity, edema, and necrosis. Axial
diffusivity (AD) tends to be sensitive to pathology, in axonal injury AD
decreases. AD of white matter tracts is reported to increase with brain
maturation. Radial diffusivity (RD) is thought to increase in white matter
during de- or dys-myelination. Despite the low inherent resolution that is
common to this modality, DTI offers an exciting new avenue for studying
alterations in human brain architecture after learning a new skill. “Diffusion
Tensor Imaging is a cutting edge imaging technique that provides quantitative
information with which to visualize and study connectivity and continuity of
neural pathways in the central and peripheral nervous systems in vivo.” (Basser
et al. 2000). It is however unknown how these changes in DTI
measures relate to the actual underlying cellular processes, and if it is a
true reflection of structural alterations in white matter due to learning or a
more transient cause like blood flow alterations. A first basic exploration of
this question was initiated by Sampaio-Baptista et al. (Sampaio-Baptista
et al. 2013). By using a rodent model they investigated how motor
skill learning would alter MRI measures of white matter and how these
alterations related to myelin basic protein (MBP) staining. They collected DTI
from rats after they trained them on a reaching task. Results showed that rats
who were trained on the reach task had significantly lower measures of density
and integrity of white matter contralateral to the reaching arm. Furthermore
MBP staining indicated that the levels of myelin were increased in the reach
group and that these levels correlated with the reach effectiveness. A critique
of this study is that they sacrificed the animals before running the MRI scan,
and only ran a between subjects analysis.

In a similar study, Sagi et al.
(Sagi, Tavor, and Assaf 2012) collected DTI scans before and after
a spatial learning task. They tested this in both human and rodent subjects and
found similar alterations, showing reduced levels of mean diffusivity in the
hippocampus. To investigate what cellular processes might underlie these alterations
they ran a histology on the rat brains and looked at microtubule associated
protein 2 (MAP-2), synaptophysin, glial fibrillary acidic protein (GFAP) and
brain derived neurotrophic factor (BDNF). They found significant increases in
the levels of synaptophysin, GFAP and BDNF in the animals that were trained on
the spatial task, but not the control animals. Indicating that alterations in
MD values after training are potentially due to changes in the number of
synaptic vesicles and/or glia density.

These types of studies are just
beginning to explore the underlying cellular mechanisms that cause change in
DTI measures after learning new skills. In this review I will highlight a
number of studies that are in support of the thesis that dynamic myelination is
necessary for effective skill learning and potentially even complex cognitive
functions. I furthermore ask the question how these behaviors are translated
and communicated to the oligodendrocytes and their precursors. Specifically,
what causes these cells to start dynamically altering migration and
differentiation, to cause myelinogenesis of axons in the white matter pathways
that ultimately lead to enhanced learning. This review aims to take a closer
look at how and when dynamic myelination occurs from early development into
adulthood.

Figure 1: Overview
of how alterations in diffusion tensor imaging measures can related to
microstructural alterations.

Myelin Development and Maintenance

The uniform and periodic spacing of myelin along the length
of axons is dependent on the organized and dynamic distribution of
oligodendrocytes and their progenitors. Until a number of years ago it was
unknown what determines this spacing, as it could be brought on by signaling
cues from axons, or OPCs could have an internal system of determining
distribution. In order to understand how OPCs dynamically altered their myelin
distribution along target axons, Kirby et al.(Kirby et al.
2006) used in vivo time lapse confocal fluorescent microscopy to
investigate OPCs surrounding axons in the spinal cord of developing zebrafish.
They created transgenic zebrafish that would express membrane-tethered
fluorescence in oligodendrocyte lineage cells using Nkx2. They found that the
remodeling and migration behavior of OPCs is highly variable, their
filopodium-like processes often migrate for many hours before myelination
occurs. Furthermore, OPCs respond to contact with neighboring OPCs by
retracting their filopodia, and will dynamically divide if they find unoccupied
spaces in order to migrate to the new location.

Although Kirby at al. showed
evidence for the dynamic behavior of OPCs in developing zebrafish spinal cord,
it remained unknown if this generalized to the cortex, would continue into
adulthood and generalized to mammal species. To answer these questions Hughes et
al.(Hughes et al. 2013) investigated how OPCs in the
cortex of adult mice maintain a homeostatic distribution even in the face of
sudden cell death. In order to do this the authors developed a mouse line that
exhibited fluorescence in cells that expressed NG2+, a marker for OPCs. They
then used two-photon imaging through a cranial window to investigate how these
cells migrated in vivo. They confirmed previous results, and showed that NG2+
containing OPCs were highly dynamic, had active filopodia that would survey the
surrounding area, while avoiding neighboring OPCs. Furthermore, they found
that, when existing cells were ablated, NG2+ cells would divide and/or migrate
to replace the damaged cells. Together these studies confirmed that OPCs are
dynamic from early development into adulthood and can actively respond to
injury by replacing damaged cells.

Neural Activity Drives Myelination

Only a selection of all axons are covered in myelin.
Cultured axons in vitro get myelinated when their diameter is bigger than 0.4µm
(Hines et al. 2015). Is this information sufficient for
oligodendrocytes in vivo, or are additional processes at work? A prerequisite
for the thesis that myelinating oligodendrocytes are essential for learning, is
that neurons and axons are capable of signaling activity levels to neighboring
oligodendrocytes and their progenitors. In this seminal paper Demerens et al.
(Demerens et al. 1996) are the first to investigate the
role of electric activity on myelin formation. They used both an in vitro and
an in vivo model to investigate if blocking or increasing action potentials
would alter axon myelination. They show that treating in vitro embryonic mouse
brains at 8 DIV for 2, 4 or 6 days with tetrodotoxin (TTX) leads up to 98%
reduction in myelinated fibers two weeks later, although this effect
disappeared at 18 DIV. Conversely a-scorpion toxin (a-ScTX), which increases
the duration and frequency of spontaneous action potentials lead to a 2.4 fold
greater number of myelinated segments. To confirm these effects in vivo they
injected TTX intravitreously in mice at day P4, after which they examined the
optic nerve at P6. It showed a reduction in myelinating oligodendrocytes of
75%, this effect disappeared when TTX was injected at P5. These are the first
indications of an interaction between neuron activity and myelination. Although
in this case the effect was very sensitive to a specific developmental time
period.

Demerens et al.(Demerens
et al. 1996) effectively showed that electric activity promotes
axonal myelination in development. What processes are at work to convert action
potentials in an axon to increased myelination by oligodendrocytes? Hines et
al. (Hines et al. 2015) used in vivo confocal
time-lapse imaging to investigate axons in the spinal cord of developing
zebrafish. They were able to label both the axons and the myelin sheets using
enhanced green labeled phox2b+-cells and red fluorescent sox10 +-cells
respectively. As seen before, exposure to TTX lead to reduced levels of myelin
wrapping, but an indiscriminate increase in action potentials due to
veratridine did not alter myelin levels. To test if selective alterations in
neural activity would bias myelination they designed a transgenic zebrafish
line with a mosaic overexpression of tetanus neurotoxin light chain (TeNT)
which inhibited synaptic vesicle exocytosis selectively in phox2b+ axons. They found
that after initial sheathing only a proportion (25%) stabilized while the rest
were retracted in an activity and vesicle release dependent fashion. These
studies show that both electric activity and vesicle release are able to signal
myelin producing cells to alter myelinogenesis in response to these signals.

Motor Learning Requires Dynamic Myelination

So far we have seen that myelin is dynamically distributed
along axons throughout adulthood and that oligodendrocytes can modify their
behavior in response to electric activity and vesicle release of axons. These
results hint at the possibility that myelin formation can take place in
response to specific behavior. Is this however sufficient for learning complex
motor behaviors? In this study Gibson et al. (Gibson et al.
2014), elegantly explore if optogenetic stimulation of the premotor
cortex in awake mice would lead to altered levels of myelination in those
regions, and if those levels of myelination were associated with improved motor
activity in the previously stimulated limb. They aimed to see if they could
induce differentiation of oligodendrocyte progenitor cells and directly
influence myelinogenesis in adult living mice. To investigate this they used a
mouse-line with channelrhodopsin infection of Thy1 expressing neural cells that
predominantly are located in layer 4 of the premotor cortex. Placement of the
optical fiber just below the pial surface allowed for stimulation of layer 4
without causing damage to those neurons. The mice would exhibit circular
walking motions during repeated unilateral stimulation at rates that were
comparable to physiological firing rates of projection neurons in this layer.
They found that both juvenile and adult mice showed altered OPC production and
myelination levels after stimulation as compared to wild type litter mates. The
administration of the proliferation marker and thymidine analog 5-ethynyl-2′-deoxyuridine (EdU) after optogenetic
activation showed labeled cells in the targeted premotor cortex and in the
subcortical white matter of the corpus callosum 3 hours after stimulation, but
not 3 weeks after stimulation. Characterization of these proliferating cells
showed that 54% labeled positive for Olig2 markers, and 24.5% for PDGF receptor-α.
To test if these results were not due to indiscriminant activity they induced a
motor seizure which did not replicate the previous results. Furthermore,
histone modifications in EdU positive cells were consistent with an alteration
in repressive and activating regulatory elements between 3-24 hours after
activation. Transmission electron microscopy results confirmed that myelin
thickness relative to axon caliber (g-ratio) was decreased, indicating an
increase in myelin thickness in the premotor cortex. This was further confirmed
with MBP staining in this region. Analysis of movement 4 weeks after neural
activation showed that these mice had improved swing speed during normal gait.
Indicating a correlation between oligodendrogenesis, myelin thickness and
movement quality. However, this improvement in muscle movement could be due to
synaptic alterations. To test this hypothesis they used an HDAC inhibitor that
would enhance synaptic plasticity, but block oligodendrogenesis. The result
showed that there was no altered g-ratio nor was there a behavioral effect
after stimulation, supporting the idea that oligodendrocyte differentiation
and/or myelination was necessary for the motor improvement after stimulation.

The usage of an HDAC blocker is a
rather crude method of inhibiting oligodendrogenesis. To combat this McKenzie et
al.(Mckenzie et al. 2014) use a conditional knock-out
of myelin regulatory factor. The authors first show that learning a new motor
skill, in this case the complex running wheel, can temporarily upregulate OPC
proliferation and differentiation in adulthood. This indicates a correlation
between motor learning and OPCs, but it is not sufficient to claim that OPC
proliferation and differentiation is necessary for motor learning. To
investigate if late born oligodendrocytes can contribute to motor learning this
group designed a mouse model where myelin regulatory factor (MyRF)
transcription in OPCs was conditionally knocked-out using the Cre-flox
technique in cells positive for the OPC-specific marker PDGF receptor-α.
Inactivation of MyRF at postnatal day 60 lead to a large reduction in immature
oligodendrocytes to about 10% of regular amounts. This reduction will inhibit
the maturation of oligodendrocytes and subsequent axonal myelination.
Administration of the proliferation marker EdU, confirmed that MyRF negative
mice had fewer new oligodendrocytes. Although this knock-out inhibited the
development of new oligodendrocytes, it did not influence the existing number
or density of myelinated axons, furthermore new oligodendrocytes were not
necessary to recall a previously learned skill. It did however alter learning
of the complex running wheel in MyRF negative mice, and it significantly
reduced the maximum speed that MyRF negative mice could accomplish in the
complex running wheel. Indicating that there is a causal relationship between oligodendrocyte
proliferation, myelination and the efficacy at which these adult mice are able
to learn a new motor skill.

These studies showed compelling
evidence for the requirement of active myelination for motor learning. A question
even the previous studies left unanswered is how exactly oligodendrocytes
receive the signal to start myelination. De Biase et al.(De Biase
et al. 2011) attempted to answer this questions by looking the
glutamate receptor NMDA. Since NMDA receptors are of great importance for
neural progenitor differentiation and migration he authors decided to
investigate what the role of NMDAR is in OPCs. They specifically explored if an
OPC specific deletion of the NMDA receptor subunit NR1 lead to an alteration in
OPC proliferation, differentiation and migration in mice. They found that this
removal did not alter survival, proliferation, migration or differentiation of
OPCs during post-natal development, and myelination was preserved. There was
however an increase in the surface expression of AMPA receptors in OPCs.

Discussion

In this overview we looked to see if adaptive axonal
myelination is necessary for learning complex motor behaviors. To explore this
thesis we first had to investigate if oligodendrocyte precursors are capable of
dynamic behavior to ensure effective development and maintenance of myelination
from embryo into adulthood. An early study in this field showed that OPCs
indeed exhibit dynamic behaviors in response to their surroundings and
neighboring OPCs. They do this to ensure an organized distribution of OPCs
along the length of the axon. Furthermore, ablation of OPCs lead to alterations
in behaviors in OPCs, e.g. migration and differentiation, to ensure the
continued coverage of axons with myelin.

Next we investigated if
oligodendrocytes and their progenitors can dynamically respond to neuronal
activity. Specifically does neural activity drive myelination, and how does it
do so. First we saw that inhibition and over-activation of action potentials in
neurons lead to altered levels of myelinogenesis. Next we saw that the
inhibition of vesicle release cause neurons to retract previously myelinated
axons. Together these results indicated that indeed neuronal activity leads to
altered myelin levels in vivo, and this is brought on by altered OPC
differentiation to cause new oligodendrocytes to myelinate axons in an activity
dependent matter.

Finally, we investigated if dynamic myelination is necessary
for learning. We first saw that optogenetic stimulation of the premotor cortex
lead to altered levels of myelin and improved motor behaviors. When
differentiation was blocked with an HDAC inhibitor myelin levels no longer
changed, and neither did the motor behaviors improve. In the next study the
took this further and saw that knock-out of myelin regulatory factor in
precursor cells prevented mice from effectively mastering the complex running
wheel. Although there must be a mechanism by which axons signal activity to
oligodendrocytes this is not due to NMDA receptor, since inactivating this
receptor did not lead to altered levels of myelination. Overall these results
indicate that indeed there is compelling evidence that dynamic myelination in
adulthood is necessary for effectively learning novel motor skills. Thus
lending support to the initial MRI studies that showed alterations in white
matter structure after motor learning took place.

Although we do not yet know what
the exact signaling mechanism is between neurons and oligodendrocytes, we can
speculate. Geurts et al.(Geurts et al. 2003)
investigated white matter of individuals with multiple sclerosis, a disease
with profound effects on myelination - specifically de-myelination, and found
that metabotropic glutamate receptor expression was altered in affected tissue.
According to Gallo and Ghiani (Gallo and Ghiani 2000) a potential
role for both metabotropic and ionotropic glutamate receptors in glia includes
regulation of proliferation and differentiation. While others (Maldonado
and Angulo 2014)suggest a role for GABA signaling.

In conclusion, the importance of
adaptive myelination of specific white matter pathways in response to learning
behaviors has given us a new appreciation for the role of oligodendrocytes and
their progenitors. Although the exact mechanism of communication between active
neurons and oligodendrocytes is yet unknown, this is yet another example of the
versatility and importance of glia cells in the brain.

Demerens, C, B Stankoff, M Logak, P Anglade, B Allinquant, F
Couraud, B Zalc, and C Lubetzki. 1996. “Induction of Myelination in the Central
Nervous System by Electrical Activity.” Proceedings of the National Academy
of Sciences of the United States of America 93 (September): 9887–92.
doi:10.1073/pnas.93.18.9887.

Gallo, V, and C a Ghiani. 2000. “Glutamate Receptors in Glla: New
Cells, New Inpute and New Functions.” Trends in Pharmacological Sciences
21 (July): 252–58.